A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit

Abstract

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+ and Ni2+ support only a single methyl transfer. MT1 homologs exist within the “GNAT” (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT didomain with bound Mn2+, malonate and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologs. These results afford an expanded understanding of MT1-GNAT structure and activity, and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

The Metabolome of a Cyanobacterial Bloom Visualized by MS/MS-Based Molecular Networking Reveals New Neurotoxic Smenamide Analogs (C, D, and E)

“…Conceivably, it may derive from an S -adenosylmethionine (SAM)-mediated methylation of an acetate precursor, the proposed first building block in the production of these smenamide-type natural products. This would be a similar biosynthetic transformation to that involved in producing the t -butyl group in apratoxin A which employs a combination of two SAM methyl transferases to incorporate these methyl groups (Grindberg et al, 2011 ; Skiba et al, 2017 ).…”

Chimeric 6‐methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity

“…Although 6MSAS fused with the MT domain, 6MSA was observed suggesting that the domain did not alter the biosynthetic pathway of the iterative PKS. AprA, a PKS involved in the biosynthesis of apratoxin A, contains two MT domains, MT1 and MT2 (Skiba et al ., ). MT1 performs two methylations to produce Me2Mal‐ACP using Mal‐ACP as the substrate.…”

Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis

“…MeMal-ACP or dimethylmalonyl (Me 2 Mal)-ACP ( Figure 2H) (Skiba et al, 2017). We determined that the carboxylate of Mal-ACP is essential for methyl transfer activity, calling into question the presumed coupling of the acyl transfer and decarboxylation activities of GNAT-like domains.…”

“…This was demonstrated in the saxitoxin pathway, where the SxtA GNAT-like domain (Figures 2C and 2D) also displayed dual activities, including a very weak acyl transfer activity and a decarboxylation activity with preference for methylmalonyl (MeMal)-ACP conversion to propionyl-ACP, the expected saxitoxin starter unit, over Mal-ACP (Chun et al, 2018). In contrast, in apratoxin A and bryostatin biosynthesis ( Figures 2E and 2F), the GNAT-like domain is truncated and possesses no detectable decarboxylation or acyl transfer activity (Skiba et al, 2017(Skiba et al, , 2018b. In these pathways, a specialized methyltransferase domain catalyzes decarboxylation coupled to methyl transfer, and the initial acyl transfer step to commence apratoxin A biosynthesis requires an enzyme not encoded in the gene cluster such as FabD, the malonyl-acyltransferase of fatty acid biosynthesis (Skiba et al, 2018b).…”